Vaccine Plasmid Topology Monitoring by Capillary Gel Electrophoresis.
Plasmid DNA has been widely used in vaccination as well as in cell and gene therapy. It exists in multiple isoforms including supercoiled, nicked or open circular and linear forms. Regulatory agencies recommend having more than 80% of the supercoiled isoform for bulk release of plasmid products; thus it should be analyzed accordingly.The traditional analysis method for plasmid DNA is agarose gel electrophoresis. However, due to time-consuming manual sample loading, visualization, and data analysis, it has limitations in obtaining consistently quantitative results. In this short communication, we introduce a fast, sensitive, and robust plasmid analysis method using capillary electrophoresis with laserinduced fluorescence detection (CE-LIF). CE-LIF analysis of the supercoiled isoform and its open circular counterpart was completed in 20 minutes with excellent sensitivity by using a common fluorescent groove binding dye. The advantage of the method was demonstrated by the purity analysis of two large plasmids (7 kb and 10 kb). The fully automated sample loading, separation and data analysis featured enhanced assay repeatability and ease of quantitation over agarose gel electrophoresis.As a worked example, analysis of plasmid samples treated at elevated temperature during an accelerated stability test also demonstrated the applicability of CELIF to monitor plasmid degradation.
Preparation of Multiwall Carbon Nanotubes Embedded Electroconductive Multi-Microchannel Scaffolds for Neuron Growth under Electrical Stimulation.
To prepare the conductive MWCNT (multiwall carbon nanotube)-agarose scaffolds with multi-microchannel for neuron growth under electrical stimulation.The scaffolds were produced by gradient freeze and lyophilization methods. The synthesized materials were characterized by SEM and near-infrared spectroscopy, and their microstructure, swelling-deswelling, conductivity, biocompatibility, and shape memory behavior were measured. A three-dimensional culture model by implanting cells into scaffolds was built, and the behaviors of RSC96 cells on scaffolds under electrical stimulation were evaluated.The addition of MWCNT did not affect the pore composition ratio and shape memory of agarose scaffolds, but 0.025% wt MWCNT in scaffolds improved the swelling ratio and water retention at the swelling equilibrium state. Though MWCNTs in high concentration had slight effect on proliferation of RSC96 cells and PC12 cells, there was no difference that the expressions of neurofilament of RSC96 cells on scaffolds with MWCNTs of different concentration. RSC96 cells arranged better along the longitudinal axis of scaffolds and showed better adhesion on both 0.025% MWCNT-agarose scaffolds and 0.05% MWCNT-agarose scaffolds compared to other scaffolds.Agarose scaffolds with MWCNTs possessed promising applicable prospect in peripheral nerve defects.
Serum Protein Gel Agarose Electrophoresis in Captive Tigers.
Given the endangered status of tigers (Panthera tigris), the health of each individual is important and any data on blood chemistry values can provide valuable information alongside the assessment of physical condition. The nature of tigers in the wild makes it is extremely difficult to obtain biological samples from free-living subjects, therefore the values obtained from captive tigers provide very useful data. Serum protein electrophoresis is a useful tool in the diagnosis and monitoring of a number of diseases. In this study, we evaluated agarose gel serum protein electrophoresis on samples from 11 healthy captive tigers. Serum electrophoresis on all 11 tiger samples successfully separated proteins into albumin, α1, α2, β1, β2 and γ globulin fractions as in other mammals. Electrophoretic patterns were comparable in all tigers. Mean± standard deviation or median and range values obtained for each protein fraction in healthy tigers were, respectively: 3.6 ± 0.2, 0.21 (0.2-0.23), 1.2 ± 0.2, 10.7 ± 0.2, 0.4 (0.3-0.6), 1.2 (1-1.8) gr/dL. The results of this preliminary study provide the first data on serum electrophoretic patterns in tigers and may be a useful diagnostic tool in the health assessment of this endangered species.
Human Keratinocyte UVB-Protective Effects of a Low Molecular Weight Fucoidan from Sargassum horneri Purified by Step Gradient Ethanol Precipitation.
Ultraviolet B (UVB) radiation-induced oxidative skin cell damage is a major cause of photoaging. In the present study, a low molecular weight fucoidan fraction (SHC4) was obtained from Sargassum horneri by Celluclast-assisted extraction, followed by step gradient ethanol precipitation. The protective effect of SHC4 was investigated in human keratinocytes against UVB-induced oxidative stress. The purified fucoidan was characterized by Fourier-transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (NMR), agarose gel-based molecular weight analysis and monosaccharide composition analysis. SHC4 had a mean molecular weight of 60 kDa, with 37.43% fucose and 28.01 ± 0.50% sulfate content. The structure was mainly composed of α-L-Fucp-(1→4) linked fucose units. SHC4 treatment dose-dependently reduced intracellular reactive oxygen species (ROS) levels and increased the cell viability of UVB exposed HaCaT keratinocytes. Moreover, SHC4 dose-dependently inhibited UVB-induced apoptotic body formation, sub-G1 accumulation of cells and DNA damage. Inhibition of apoptosis was mediated via the mitochondria-mediated pathway, re-establishing the loss of mitochondrial membrane potential. The UVB protective effect of SHC4 was facilitated by enhancing intracellular antioxidant defense via nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling. Further studies may promote the use of SHC4 as an active ingredient in cosmetics and nutricosmetics.
IKZF1 Deletions as a Prognostic Factor in Costa Rican Patients With Pediatric B-Cell Acute Lymphoblastic Leukemia.
The IKZF1 gene encodes for Ikaros, a transcriptional factor in B-cell development. Deletions in this gene have been associated with a worse prognosis in B-cell acute lymphoblastic leukemia (B-ALL). We evaluated the presence of these alterations in all Costa Rican pediatric patients diagnosed with B-ALL between 2011 and 2014, treated with a modified Berlin-Frankfurt-Münster therapeutic protocol. Multiplex polymerase chain reaction with 2 detection methods (agarose gel and gene scanning) was used to detect intragenic deletions and multiplex ligation-dependent probe amplification for whole-gene deletions. Differences between groups (normal vs. deleted IKZF1) were analyzed by the χ test, the Kaplan-Meier test was used to calculate relapse-free survival and overall survival, and Cox regression was performed for multivariant analysis. Minimum follow-up was 4.5 years. Incidence of IKZF1 deletions was 12.9% (n=20), with an equal amount of intragenic and complete gene deletions. Adverse karyotype (P=0.048), high-risk category (P=0.030), occurrence of relapse (P=0.021), and medullar relapse (P=0.011) were statistically associated with the presence of deletions in IKZF1. Relapse-free survival at 54 months was lower in patients harboring an IKZF1 deletion than that in patients with IKZF1-wt (40.0% vs. 66.7%; P=0.014). Patients with B-ALL and IKZF1 deletions, showed a poorer relapse-free survival, in comparison with patients with IKZF1-wt, suggesting that IKZF1 status is an independent prognostic factor for pediatric patients with B-ALL.
An enzyme inhibition-based lab-in-a-syringe device for point-of-need determination of pesticides.
An enzyme inhibition-based lab-in-a-syringe (EI-LIS) device was developed by integrating a 1-naphthol-linked bi-enzymatic reaction (sensor core) into the LIS (sensor device) for point-of-need monitoring of pesticide residues. The integration relies on the rational design of two reaction pads. The conjugate pad is a polyester fiber membrane loaded with plant-esterase, an alternative to acetylcholinesterase. Besides pesticide capture, plant-esterase also mediates the hydrolysis of 1-naphthyl acetate, generating 1-naphthol. The detection pad is an agarose gel entrapping oxidized 3,3′,5,5′-tetramethylbenzidine (oxTMB) from Fe(iii) meso-tetra(N-methyl-4-pyridyl) porphyrin (FeTMPyP4)-catalyzed TMB oxidation. Both pads were embedded into their cartridges and then connected to a syringe. Under syringe pumping, 1-naphthol vertically flowed from the conjugate to the detection cartridge, linking the two pads. If plant-esterase was intact, 1-naphthol would reduce oxTMB, causing a color change of the detection pad from blue to colorless. If the plant-esterase activity was inhibited by pesticides, less 1-naphthol was produced, and the blue color of the detection pad would be partially or wholly retained. The deeper the blue color, the greater the pesticide concentration. This chromogenic pattern is responsible for a highly sensitive readout (detection limits of dichlorvos: 0.1 nM with the naked eye and 0.07 nM with a spectrometer).
CircSAMD4A regulates cell progression and epithelial‑mesenchymal transition by sponging miR‑342‑3p via the regulation of FZD7 expression in osteosarcoma.
Osteosarcoma (OS) is a primary malignant tumor with a complex etiology. Therefore, research into the pathogenesis of osteosarcoma is considered a priority. Circular RNAs play important roles in cell metabolism and in the immune response and are closely associated with cancer treatment. However, research into the association of circular RNAs with osteosarcoma is limited. In the present study, CircSAMD4A was validated by RT‑qPCR and agarose gel electrophoresis. CircSAMD4A and miR‑342‑3p expression was detected by RT‑qPCR. The relative protein expression levels were measured by western blot analysis. MTT assay and flow cytometry were used to detect cell cytotoxicity and apoptosis, respectively. Transwell assay was applied to assess cell migration and invasion. Dual‑luciferase reporter assay was used to determine the association among CircSAMD4A, Frizzled‑7 (FZD7) and miR‑342‑3p. In vivo, subcutaneous tumor formation assay was performed in an experiment with nude mice. The results revealed that the expression levels of CircSAMD4A and FZD7 were upregulated, while those of miR‑342‑3p were downregulated in OS tissues and cells. The inhibition of CircSAMD4A suppressed cell progression and epithelial‑mesenchymal transition (EMT), and promoted cell apoptosis in OS. The reduction of miR‑342‑3p reversed the effects of CircSAMD4A downregulation on cell cytotoxicity, migration, invasion, apoptosis and EMT in OS, while FZD7 overexpression blocked the effect of miR‑342‑3p upregulation on OS progression. The suppressive effect of sh‑CircSAMD4A on tumor growth was thus verified in OS. Overall, the present study demonstrated that CircSAMD4A affected cell cytotoxicity, invasion, apoptosis, migration and EMT by regulating the miR‑342‑3p/FDZ7 axis in OS, thereby providing a novel regulatory mechanism and a potential therapeutic target for OS.
Injectable Hydrogel for NIR-II Photo-Thermal Tumor Therapy and Dihydroartemisinin-Mediated Chemodynamic Therapy.
In traditional Chinese medicine, dihydroartemisinin (DHA) is the focus of extensive attention because of its unique activity with Fe2+ to produce reactive oxygen species (ROS) and promote apoptosis. In this work, we designed a newfangled ink@hydrogel containing FeCl3, traditional Chinese ink (Hu Kaiwen ink), and agarose hydrogel to create a synergistic activity with DHA in the treatment of cancer. When the system is irradiated under 1,064 nm for a few minutes, the ink in the ink@hydrogel converts the light to heat and hyperthermia causes the reversible hydrolysis of hydrogel. Then, Fe3+ quickly diffuses from the hydrogel to the tumor microenvironment and is reduced to Fe2+ to break the endoperoxide bridge in pre-injected DHA, which results in the release of free radicals for a potent anticancer action. To our knowledge, this is the first report of a hydrogel tumor therapy system that induces a photo-thermal response in the second near infrared window (NIR-II). in vivo experiments also showed a significant effect of DHA-Fe2+ in chemodynamic therapy (CDT) and in photo-thermal therapy. This hydrogel platform provided an encouraging idea for synergistic tumor therapy.
A dual-stimulation strategy in a micro-chip for the investigation of mechanical associative learning behavior of C. elegans.
During the past decades, few micro-devices for analysis of associative learning behavior have been reported. In this work, an agarose-PDMS hybridized micro-chip was developed to establish a new associative learning model between mechanosensation and food reward in C. elegans. The micro-chip consisted of column arrays which mimicked mechanical stimulation to C. elegans. After trained by pairing bacterial food and mechanical stimuli in the chip, the worms exhibited associative learning behavior and gathered in the regions where there was food during training. The key research findings include: (1) Associative learning behavior of C. elegans could be generated and quantitatively analyzed by this developed micro-chip. (2) Associative learning behavior could be enhanced by extending the training time and developmental stage. (3) Mechanosensation-related genes and neurotransmitters signals had effects on the learning behavior. (4) The associative learning ability could be strengthened by exogenous dopamine in both wild type and mutants. We validated that the design of the micro-chip was useful and convenient for the study of learning behavior based on mechanosensation.
High SOX8 expression promotes tumor growth and predicts poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma.
According to our previous study, GOLPH3 is markedly up-expressed in tongue squamous cell carcinoma (TSCC), which is also associated with poor survival. However, it remains unclear about key upstream and downstream mechanisms of GOLPH3. This study aimed to illuminate new mechanisms modulating GOLPH3 upregulation and promoting TSCC development at the molecular level. Using mass spectrometry and agarose-streptavidin-biotin pull-down analyses, SOX8 (SRY-Box 8) was identified to be the new protein to bind the GOLPH3 promoter.